Growth differentiation factor-9

ABSTRACT

Growth differentiation factor-9 (GDF-9) is disclosed along with its polynucleotide sequence and amino acid sequence. Also disclosed are diagnostic and therapeutic methods of using the GDF-9 polypeptide and polynucleotide sequences.

This is a continuation of U.S. application Ser. No. 09/172,062, filedOct. 13, 1998, now U.S. Pat. No. 6,191,261 which is a divisional of U.S.application Ser. No. 08/491,835, filed Oct. 23, 1995, now U.S. Pat. No.5,821,056, which is a 371 application of PCT/US 94/00685, filed Jan. 12,1994, which is a continuation-in part application of U.S. Ser. No.08/003,303, filed Jan. 12, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-9 (GDF-9).

2. Description of Related Art

The transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis,myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions of the proteins are all structurallyrelated and the different family members can be classified into distinctsubgroups based on the extent of their homology. Although the homologieswithin particular subgroups range from 70% to 90% amino acid sequenceidentity, the homologies between subgroups are significantly lower,generally ranging from only 20% to 50%. In each case, the active speciesappears to be a disulfide-linked dimer of C-terminal fragments. For mostof the family members that have been studied, the homodimeric specieshas been found to be biologically active, but for other family members,like the inhibins (Ling, et al., Nature, 321:779, 1986) and the TGF-βs(Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also beendetected, and these appear to have different biological properties thanthe respective homodimers.

The inhibins and activins were originally purified from follicular fluidand shown to have counteracting effects on the release offollicle-stimulating hormone by the pituitary gland. Although the mRNAsfor all three inhibin/activin subunits (αa, βA and βB) have beendetected in the ovary, none of these appear to be ovary-specific(Meunier, et al, Proc.Natl.Acad.Sci. USA, 85:247, 1988). MIS has alsobeen shown to be expressed by granulosa cells and the effects of MIS onovarian development have been documented both in vivo in transgenic miceexpressing MIS ectopically (Behringer, supra) and in vitro in organculture (Vigier, et al., Development, 100:43, 1987).

Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-9, a polynucleotide sequence which encodes the factor and antibodieswhich are immunoreactive with the factor. This factor appears to relateto various cell proliferative disorders, especially those involvingovarian tumors, such as granulosa cell tumors.

Thus, in one embodiment, the invention provides a method for detecting acell proliferative disorder of ovarian origin and which is associatedwith GDF-9. In another embodiment, the invention provides a method oftreating a cell proliferative disorder associated with abnormal levelsof expression of GDF-9, by suppressing or enhancing GDF-9 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of GDF-9 mRNA in adult tissues.

FIGS. 2a-c show nucleotide and predicted amino acid sequence of murineGDF-9 (SEQ ID NO:3 and SEQ ID NO:4, respectively). ConsensusN-glycosylation signals are denoted by plain boxes. The putativetetrabasic processing sites are denoted by stippled boxes. The in-frametermination codons upstream of the putative initiating ATG and theconsensus poyadenylation signals are underlined. The poly A tails arenot shown. Numbers indicate nucleotide position relative to the 5′ end.

FIGS. 3a and 3 b show the alignment of the C-terminal sequences of GDF-9with other membranes of the TGF-β family (SEQ ID NO:5-25). The conservedcysteine residues are shaded. Dashes denote gaps introduced in order tomaximize alignment.

FIGS. 4a and 4B show amino acid homologies among the different membersof the TGF-β superfamily. Numbers represent percent amino acididentities between each pair calculated from the first conservedcysteine to the C-terminus. Boxes represent homologies amonghighly-related members within particular subgroups.

FIG. 5 shows the immunohistochemical localization of GDF-9 protein.Adjacent sections of an adult ovary were either stained with hematoxylinand eosin (FIG. 5a) or incubated with immune (FIG. 5b) or pre-immune(FIG. 5c) serum at a dilution of 1:500. Anti-GDF-9 antiserum wasprepared by expressing the C-terminal portion of murine GDF-9 (residues308-441) in bacteria, excising GDF-9 protein from preparative SDS gels,and immunizing rabbits. Sites of antibody binding were visualized usingthe Vectastain ABC kit (Vector Labs).

FIG. 6 shows a comparison of the predicted amino acid sequences ofmurine (top lines) and human (bottom lines) GDF-9 (SEQ ID NO:4 and SEQID NO:26, respectively). Numbers represent amino acid positions relativeto N-termini. Vertical lines represent sequence identities. Dotsrepresent gaps introduced in order to maximize the alignment. The clearbox shows the predicted proteolytic processing sites. The shaded boxesshow the cysteine residues in the mature region of the proteins. Thebars at the bottom show a schematic of the pre-(clear) and mature(shaded) regions of GDF-9 with the percent sequence identities betweenthe murine and human sequences shown below.

FIG. 7 shows in situ hybridization to adult ovary sections using GDF-9RNA probe. [³⁵S]-labeled anti-sense (FIG. 7a and 7 c) or sense (FIG. 7band 7 d) GDF-9 RNA probes were hybridized to adjacent paraffin-embeddedsections of ovaries fixed in 4% paraformaldehyde. Sections were dippedin photographic emulsion, exposed, developed, and then stained withhematoxlin and eosin. Two representative fields are shown.

FIG. 8 shows in situ hybridization toa postnatal day 4 ovary sectionusing an antisense GDF-9 RNA probe. Sections were prepared as describedfor FIG. 7. Following autoradiography and staining, the section wasphotographed under bright-field (FIG. 8a) or dark-field (FIG. 8b)illumination.

FIG. 9 shows in situ hybridization to postnatal day 8 ovary sectionsusing an antisense (FIG. 9a) or sense (FIG. 9b) GDF-9 RNA probe.Sections were prepared as described in FIG. 7.

FIG. 10 shows in situ hybridization to adult oviduct sections using anantisense (FIG. 10a) or sense (FIG. 10b) GDF-9 RNA probe. Sections wereprepared as described for FIG. 7.

FIG. 11 shows in situ hybridization to an adult oviduct (0.5 daysfollowing fertilization) section using an antisense GDF-9 RNA probe.Sections were prepared as described for FIG. 7. Followingautoradiography and staining, the section was photographed underbright-field (FIG. 11a) or dark-field (FIG. 11b) illumination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-9 and a polynucleotide sequence encoding GDF-9. Unlike other membersof the TGF-β superfamily, GDF-9 expression is highly tissue specific,being expressed in cells primarily in ovarian tissue. In one embodiment,the invention provides a method for detection of a cell proliferativedisorder of the ovary, which is associated with GDF-9 expression. Inanother embodiment, the invention provides a method for treating a cellproliferative disorder associated with abnormal expression of GDF-9 byusing an agent which suppresses or enhances GDF-9 activity.

The TGF-β superfamily consists of multifunctionaly polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-9 protein of this invention and the members of the TGF-β family,indicates that GDF-9 is a new member of the family of growth anddifferentiation factors. Based on the known activities of many of theother members, it can be expected that GDF-9 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

For example, another regulatory protein that has been found to havestructural homology with TGF-β is inhibin, a specific and potentpolypeptide inhibitor of the pituitary secretion of FSH. Inhibin hasbeen isolated from ovarian follicular fluid. Because of its suppressionof FSH, inhibin has potential to be used as a contraceptive in bothmales and females. GDF-9 may possess similar biological activity sinceit is also an ovarian specific peptide.Inhibin has also been shown to beuseful as a marker for certain ovarian tumors (Lappohn, et al., N. Engl.J. Med., 321:790, 1989). GDF-9 may also be useful as a marker foridentifying primary and metastatic neoplasms of ovarian origin.Similarly, GDF-9 may be useful as an indicator of developmentalanomalies in prenatal screening procedures.

Another peptide of the TGF-β family is MIS, produced by the testis andresponsible for the regression of the Mullerian ducts in the maleembryo. MIS has been show to inhibit the growth of human ovarian cancerin nude mice (Donahoe, et al., Ann. Surg., 194:472, 1981). GDF-9 mayfunction similarly and may, therefore, be useful as an anti-canceragent, such as for the treatment of ovarian cancer.

GDF-9 may also function as a growth stimulatory factor and, therefore,be useful for the survival of various cell populations in vitro. Inparticular, if GDF-9 plays a role in oocyte maturation, it may be usefulin in vitro fertilization procedures, e.g., in enhancing the successrate. Many of the members of the TGF-β family are also importantmediators of tissue repair. TGF-β has been shown to have marked effectson the formation of collagen and causes a striking angiogenic responsein the newborn mouse (Roberts, et al., Proc. Natl. Acad. Sci. USA,83:4167, 1966). GDF-9 may also have similar activities and may be usefulin repair of tissue injury caused by trauma or burns for example.

The term “substantially pure” as used herein refers to GDF-9 which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify GDF-9 using standard techniques for protein purification. Thesubstantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the GDF-9 polypeptide canalso be determined by amino-terminal amino acid sequence analysis. GDF-9polypeptide includes functional fragments of the polypeptide, as long asthe activity of GDF-9 remains. Smaller peptides containing thebiological activity of GDF-9 are included in the invention.

The invention provides polynucleotides encoding the GDF-9 protein. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode GDF-9.It is understood that all polynucleotides encoding all or a portion ofGDF-9 are also included herein, as long as they encode a polypeptidewith GDF-9 activity. Such polynucleotides include naturally occurring,synthetic, and intentionally manipulated polynucleotides. For example,GDF-9 polynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for GDF-9 also includes antisense sequences. Thepolynucleotides of the invention include sequences that are degenerateas a result of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention as long as the aminoacid sequence of GDF-9 polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

Specifically disclosed herein is a cDNA sequence for GDF-9 which is 1712base pairs in length and contains an open reading frame beginning with amethionine codon at nucleotide 29. The encoded polypeptide is 441 aminoacids in length with a molecular weight of about 49.6 kD, as determinedby nucleotide sequence analysis. The GDF-9 sequence contains a core ofhydrophobic amino acids near the N-terminus, suggestive of a signalsequence for secretion. GDF-9 contains four potential N-glycosylationsites at asparagine residues 163, 229, 258, and 325 and a putativetetrabasic proteolytic processing site (RRRR; SEQ ID NO: 27) at aminoacids 303-306. The mature C-terminal fragment of GDF-9 is predicted tobe 135 amino acids in length and have an unglycosylated molecular weightof about 15.6 kD, as determined by nucleotide sequence analysis. Oneskilled in the art can modify, or partially or completely remove theglycosyl groups from the GDF-9 protein using standard techniques.Therefore, the functional protein or fragments thereof of the inventionincludes glycosylated, partially glycosylated and unglycosylated speciesof GDF-9.

The degree of sequence identity of GDF-9 with known TGF-β family membersranges from a minimum of 21% with Mullerian inhibiting substance (MIS)to a maximum of 34% with bone morphogenetic protein-4 (BMP-4). GDF-9specifically disclosed herein differs from the known family members inits pattern of cysteine residues in the C-terminal region. GDF-9 lacksthe fourth cysteine of the seven cysteines present in other familymembers; in place of cysteine at this position, the GDF-9 sequencecontains a serine residue. This GDF-9 does not contain a seventhcysteine residue elsewhere in the C-terminal region.

Minor modifications of the recombinant GDF-9 primary amino acid sequencemay result in proteins which have substantially equivalent activity ascompared to the GDF-9 polypeptide described herein. Such modificationsmay be deliberate, as by site-directed mutagenesis, or may bespontaneous. All of the polypeptides produced by these modifications areincluded herein as long as the biological activity of GDF-9 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-9 biological activity.

The nucleotide sequence encoding the GDF-9 polypeptide of the inventionincludes the disclosed sequence and conservative variations thereof. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine. glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences and 2) antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures.

Preferably the GDF-9 polynucleotide of the invention is derived from amammalian organism, and most preferably from a mouse, rat, or human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, 1981).

The development of specific DNA sequences encoding GDF-9 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-9 peptides having at least one epitope, usingantibodies specific for GDF-9. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-9 cDNA.

DNA sequences encoding GDF-9 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the GDF-9 polynucleotide sequences may beinserted into a recombinant expression vector, The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-9 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene ,125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-9 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-9 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors. ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention includes antibodies immunoreactive with GDF-9 polypeptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂, which are capable of binding an epitopic determinanton GDF-9.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. The GDF-9polynucleotide that is an antisense molecule is useful in treatingmalignancies of the various organ systems, particularly, for example,the ovaries. Essentially, any disorder which is etiologically linked toaltered expression of GDF-9 could be considered susceptible to treatmentwith a GDF-9 suppressing reagent.

The invention provides a method for detecting a cell proliferativedisorder of the ovary which comprises contacting an anti-GDF-9 antibodywith a cell suspected of having a GDF-9 associated disorder anddetecting binding to the antibody. The antibody reactive with GDF-9 islabeled with a compound which allows detection of binding to GDF-9. Forpurposes of the invention, an antibody specific for GDF-9 polypeptidemay be used to detect the level of GDF-9 in biological fluids andtissues. Any specimen containing a detectable amount of antigen can beused. A preferred sample of this invention is tissue of ovarian origin,specifically tissue containing granulosa cells or ovarian follicularfluid. The level of GDF-9 in the suspect cell can be compared with thelevel in a normal cell to determine whether the subject has aGDF-9-associated cell proliferative disorder. Preferably the subject ishuman.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene. polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radio-isotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in viva diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-9-associated diseasein a subject. Thus, for example, by measuring the increase or decreasein the number of cells expressing antigen comprising a polypeptide ofthe invention or changes in the concentration of such antigen present invarious body fluids, it would be possible to determine whether aparticular therapeutic regimen aimed at ameliorating theGDF-9-associated disease is effective. The term “ameliorate” denotes alessening of the detrimental effect of the GDF-9-associated disease inthe subject receiving therapy.

The present invention identifies a nucleotide sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore, it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of GDF-9,nucleic acid sequences that interfere with GDF-9 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-9 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target GDF-9-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

Ribazymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that., because they are sequence-specific, onlymRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 34:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders which are mediated by GDF-9 protein. Suchtherapy would achieve its therapeutic effect by introduction of theGDF-9 antisense polynucleotide into cells having the proliferativedisorder. Delivery of antisense GDF-9 polynucleotide can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system.

Especially preferred for therapeutic delivery of antisense sequences isthe use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a GDF-9 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the GDF-9 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to 2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-9 antisense polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Uposomes are artificial membrane vesicles whichare useful as delivery vehicles in vitro and in vivo. It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-4.0μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the expression of GDF-9 in the reproductive tract, there are avariety of applications using the polypeptide, polynucleotide andantibodies of the invention, related to contraception, fertility andpregnancy. GDF-9 could play a role in regulation of the menstrual cycleand, therefore, could be useful in various contraceptive regimens.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

To identify a new member of the TGF-β superfamily, degenerateoligonucleotides were designed which corresponded to two conservedregions among the known family members: one region spanning the twotryptophan residues conserved in all family members except MIS and theother region spanning the invariant cysteine residues near theC-terminus. These primers were used for polymerase chain reactions onmouse genomic DNA followed by subcloning the PCR products usingrestriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate known members of the superfamily.

GDF-9 was identified from a mixture of PCR products obtained with theprimers SJL160 (5′-CCGGAATTCGGITGG(G/C/A)A(G/A/T/C)(G/C/A)A(G/A/T/C)TGG(A/G)TI(A/G)TI(T/G)CICC-3′) (SEQUENCE ID NO. 1) and SJL153(5′-CCGGAATTC(A/G)CAI(G/C)C(A/G)CAIC(T/C)(G/A/T-IC)(C/G/T)TIG(T/C)I(G/A)(T/C)CAT-3′)(SEQUENCE ID NO. 2). PCR using these primers was carried out with 2 μgmouse genomic DNA at 94° C. for 1 min, 50° C. for 2 min, and 72° C. for2 min for 40 cycles.

PCR products of approximately 280 bp were gel-purified, digested withEco RI, gel-purified again, and subcloned in the Bluescript vector(Stratagene, San Diego, Calif.). Bacterial colonies carrying individualsubclones were picked into 96 well microtiter plates, and multiplereplicas were prepared by plating the cells onto nitrocellulose. Thereplicate filters were hybridized to probes representing known membersof the family, and DNA was prepared from non-hybridizing colonies forsequence analysis.

The primer combination of SJL160 and SJL153, yielded three knownsequences (inhibin βB, BMP-2, and BMP-4) and one novel sequence(designated GDF-9) among 145 subclones analyzed.

RNA isolation and Northern analysis were carried out as describedpreviously (Lee,S. J., Mol. Endocrinol. 4:1034, 1990). An oligodT-primed cDNA library was prepared from 2.5-3 μg of ovary polyA-selected RNA in the lambda ZAP II vector according to the instructionsprovided by Stratagene. The ovary library was not amplified prior toscreening. Filters were hybridized as described previously (Lee, S.-J.,Proc. Natl. Acad. Sci. USA., 88:4250-4254, 1991). DNA sequencing of bothstrands was carried out using the dideoxy chain termination method(Sanger, et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467, 1977) and acombination of the S1 nuclease/exonuclease III strategy (Henikoff, S.,Gene, 28:351-359, 1984) and synthetic oligonucleotide primers.

EXAMPLE 2 Expression Pattern and Sequence of GDF-9

To determine the expression pattern of GDF-9, RNA samples prepared froma variety of adult tissues were screened by Northern analysis. Fivemicrograms of twice polyA-selected RNA prepared from each tissue wereelectrophoresed on formaldehyde gels, blotted and probed with GDF-9. Asshown in FIG. 1, the GDF-9 probe detected a 1.7 kb mRNA expressedexclusively in the ovary.

A mouse ovary cDNA library of 1.5×10⁶ recombinant phage was constructedin lambda ZAP II and screened with a probe derived from the GDF-9 PCRproduct. The nucleotide sequence of the longest of nineteen hybridizingclones is shown in FIG. 2. Consensus N-glycosylation signals are denotedby plain boxes. The putative tetrabasic processing sites are denoted bystippled boxes. The in-frame termination codons upstream of the putativeinitiating ATG and the consensus polyadenylation signals are underlined.The poly A tails are not shown. Numbers indicate nucleotide positionrelative to the 5′ end. The 1712bp sequence contains a long open readingframe beginning with a methionine codon at nucleotide 29 and potentiallyencoding a protein 441 amino acids in length with a molecular weight of49.6 kD. Like other TGF-β family members, the GDF-9 sequence contains acore of hydrophobic amino acids near the N-terminus suggestive of asignal sequence for secretion. GDF-9 contains four potentialN-glycosylation sites at asparagine residues 163, 229, 258, and 325 anda putative tetrabasic proteolytic processing site (RRRR) (amino acidresidues 303-306 of SEQ ID NO:4) at amino acids 303-306. The matureC-terminal fragment of GDF-9 is predicted to be 135 amino acids inlength and have an unglycosylated molecular weight of 15.6 kD.

Although the C-terminal portion of GDF-9 clearly shows homology with theother family members, the sequence of GDF-9 is significantly divergedfrom those of the other family members (FIGS. 3 and 4). FIG. 3 shows thealignment of the C-terminal sequences of GDF-9 with the correspondingregions of human GDF-1 (Lee, Proc. Natl. Acad. Sci. USA, 88:4250-4254,1991), Xenopus Vg-1 (Weeks, et al., Cell, 51:861-867, 1987), human Vgr-1(Celeste, et al., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), humanOP-1 (Ozkaynak, et al., EMBO J., 9:2085-2093, 1990), human BMP-5(Celeste et al., Proc. Natl. Acad. Sci. USA, 87, 9843-9847, 1990),Drosophilia 60A (Wharton, et al., Proc. Natl. Acad. Sci. USA,88:9214-9218, 1991), human BMP-2 and 4 (Wozney, et al., Science,242:1528-1534, 1988), Drosophilia DPP (Padgett, et al., Nature,325:81-84,1987), human BMP-3 (Wozney, et al., Science,242:1528-1534,1988), human MIS (Cate, et al., Cell, 45:685-698, 1986),human inhibin, βA, and βB (Mason, et al., Biochem. Biophys. Res.Commun., 135:957-964,1986), human TGF-β1 (Derynck, et al., Nature,316:701-705, 1985), human TGF-β2 (deMartin, et al., EMBO J.,6:3673-3677, 1987), human TGF-β3 (ten Dijke, et al., Proc. Natl. Acad.Sci. USA, 5:4715-4719, 1988), chicken TGF-β4 (Jakowlew, et al., Mol.Endocrinol., 2:1186-1195, 1988), and Xenopus TGF-β5 (Kondaiah, et al.,J. Biol. Chem., 265:1089-1093, 1990). The conserved cysteine residuesare shaded. Dashes denote gaps introduced in order to maximize thealignment.

FIG. 4 shows the amino acid homologies among the different members ofthe TGF-6 superfamily. Numbers represent percent amino acid identitiesbetween each pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups.

The degree of sequence identify with known family members ranges from aminimum of 21% with MIS to a maximum of 34% with BMP-4. Hence, GDF-9 iscomparable to MIS in its degree of sequence divergence from the othermembers of this superfamily. Moreover, GDF-9 shows no significantsequence homology to other family members in the pro-region of themolecule. GDF-9 also differs from the known family members in itspattern of cysteine residues in the C-terminal region. GDF-9 lacks thefourth cysteine of the seven cysteines that are present in all otherfamily members; in place of cysteine at this position, the GDF-9sequence contains a serine residue. In addition, GDF-9 does not containa seventh cysteine residue elsewhere in the C-terminal region.

EXAMPLE 3 Immunochemical Localization of GDF-9 in the Zona Pellucida

To determine whether GDF-9 mRNA was translated, sections of adultovaries were incubated with antibodies directed against recombinantGDF-9 protein. In order to raise antibodies against GDF-9, portions ofGDF-9 cDNA spanning amino acids 30 to 295 (pro-region) or 308 to 441(mature region) were cloned into the T7-based pET3 expression vector(provided by F. W. Studier, Brookhaven National Laboratory), and theresulting plasmids were transformed into the BL21 (DE3) bacterialstrain. Total cell extracts from isopropyl B-D-thiogalactoside-inducedcells were electrophoresed on SDS/polyacrylamide gels, and the GDF-9protein fragments were excised, mixed with Freund's adjuvant, and usedto immunize rabbits by standard methods known to those of skill in theart. All immunizations were carried out by Spring Valley Lab(Sykesville, Md.). The presence of GDF-9-reactive antibodies in the seraof these rabbits was assessed by Western analysis ofbacterially-expressed protein fragments. The resulting serum was shownto react with the bacterially-expressed protein by Western analysis.

For immunohistochemical studies, ovaries were removed from adult mice,fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sitesof antibody binding were detected by using the Vectastain ABC kit,according to the instructions provided by Vector Laboratories. FIG. 5shows the immunohistochemical localization of GDF-9 protein. Adjacentsections of an adult ovary were either stained with hematoxylin andeosin (FIG. 5a) or incubated with immune (FIG. 5b) or pre-immune (FIG.5c) serum at a dilution of 1:500. As shown in FIG. 5b, the antiserumdetected protein solely in oocytes. No staining was detected usingpre-immune serum (FIG. 5c). Hence, GDF-9 protein appears to translatedin vivo by oocytes.

EXAMPLE 4 Isolation of Human GDF-9

In order to isolate a cDNA clone encoding human GDF-9, a cDNA librarywas constructed in lambda ZAP II using poly A-selected RNA prepared froman adult human ovary. From this library, a cDNA clone containing theentire human GDF-9 coding sequence was identified using standard,screening techniques as in Example 1 and using the murine GDF-9 clone asa probe. A comparison of the predicted amino acid sequences of murine(top lines) and human (bottom lines) GDF-9 is shown in FIG. 6. Numbersrepresent amino acid positions relative to the N-termini. Vertical linesrepresent sequence identities. Dots represent gaps introduced in orderto maximize the alignment. The clear box shows the predicted proteolyticprocessing sites. The shaded boxes show the cysteine residues in themature region of the proteins. The bars at the bottom show a schematicof the pre-(clear) and mature (shaded) regions of GDF-9 with the percentsequence identities between the murine and human sequences shown below.

Like murine GDF-9, human GDF-9 contains a hydrophobic leader sequence, aputative RXXR(SEQ ID NO:28) proteolytic cleavage site, and a C-terminalregion containing the hallmarks of other TGF-β family members. Murineand human GDF-9 are 64% identical in the pro- region and 90% identicalin the predicted mature region of the molecule. The high degree ofhomology between the two sequences suggests that human GDF-9 plays animportant role during embryonic development and/or in the adult ovary.

EXAMPLE 5 Nucleic Acid Detection of Expression of GDF-9 in Oocytes

In order to localize the expression of GDF-9 in the ovary, in situhybridization to mouse ovary sections was carried out using an antisenseGDF-9 RNA probe. FIG. 7 shows in situ hybridization to adult ovarysections using a GDF-9 RNA probe. [³⁵S]-labeled anti-sense (FIG. 7a and7 c) or sense (FIG. 7b and 7 d) GDF-9 RNA probes were hybridized toadjacent paraffin-embedded sections of ovaries fixed in 4%paraformaldehyde. Sections were dipped in photographic emulsion,exposed, developed, and then stained with hematoxylin and eosin. Tworepresentative fields are shown.

As shown in FIGS. 7a and 7c, GDF-9 mRNA was detected primarily inoccytes in adult ovaries. Every occyte (regardless of the stage offollicular development) examined showed GDF-9 expression, and noexpression was detected in any other cell types. No hybridization wasseen using a control GDF-9 sense RNA probe (FIG. 7b and 7 d). Hence,GDF-9 expression appears to be oocyte-specific in adult ovaries.

To determine the pattern of expression of GDF-9 mRNA during ovariandevelopment, sections of neonatal ovaries were probed with a GDF-9 RNAprobe. FIG. 8 shows in situ hybridization to a postnatal day: 4 ovarysection using an antisense GDF-9 RNA probe. Sections were prepared asdescribed for FIG. 7. Following autoradiography and staining, thesection was photographed under bright-field (FIG. 8a) or dark-field(FIG. 8b) illumination.

FIG. 9 shows in situ hybridization to postnatal day 8 ovary sectionsusing an antisense (FIG. 9a) or sense (FIG. 9b) GDF-9 RNA probe.Sections were prepared as described for FIG. 7.

GDF-9 mRNA expression was first detected at the onset of folliculardevelopment. This was most clearly evident at postnatal day 4, whereonly cocytes that were present in follicles showed GDF-9 expression(FIG. 8); no expression was seen in oocytes that were not surrounded bygranulosa cells. By postnatal day 8, every oocyte appeared to haveundergone follicular development, and every oocyte showed GDF-9expression (FIG. 9).

To determine whether GDF-9 was also expressed following ovulation,sections of mouse oviducts were examined by in situ hybridization. FIG.10 shows in situ hybridization to adult oviduct sections using anantisense (FIG. 10a) or sense (FIG. 10b) GDF-9 RNA probe. Sections wereprepared as described for FIG. 7.

FIG. 11 shows in situ hybridization to an adult oviduct (0.5 daysfollowing fertilization) section using an antisense GDF-9 RNA probe.Sections were prepared as described for FIG. 7. Followingautoradiography and staining, the section was photographed underbright-field (FIG. 11a) or dark-field (FIG. 11b) illumination.

As shown in FIG. 10, GDF-9 was expressed by cocytes that had beenreleased into the oviduct. However, the expression of GDF-9 mRNA turnedoff rapidly following fertilization of the oocytes; by day 0.5 followingfertilization, only some embryos (such as the one shown in FIG. 11)expressed GDF-9 mRNA, and by day 1.5, all embryos were negative forGDF-9 expression.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleotide sequence for the primer, SJL160, forGDF-9 (page 26, lines 15 and 16);

Sequence ID No. 2 is the nucleotide sequence for the primer, SJL153, forGDF-9 (page 26, lines 17 and 18);

Sequence ID No. 3 is the nucleotide and deduced amino acid sequence forGDF-9 (FIG. 2);

Sequence ID No. 4 is the deduced amino acid sequence for GDF-9 (FIG. 2);

Sequence ID No. 5 is the amino acid sequence of the C-terminus 6f GDF-3(FIG. 3);

Sequence ID No. 6 is the amino acid sequence of the C-terminus of GDF-9(FIG. 3);

Sequence ID No. 7 is the amino acid sequence of the C-terminus of GDF-1(FIG. 3);

Sequence ID No. 8 is the amino acid sequence of the C-terminus of Vg-1(FIG. 3);

Sequence ID No. 9 is the amino acid sequence of the C-terminus of Vgr-1(FIG. 3);

Sequence ID No. 10 is the amino acid sequence of the C-terminus of OP-1(FIG. 3);

Sequence ID No. 11 is the amino acid sequence of the C-terminus of BMP-5(FIG. 3);

Sequence ID No. 12 is the amino acid sequence of the C-terminus of 60A(FIG. 3);

Sequence ID No. 13 is the amino acid sequence of the C-terminus of BMP-2(FIG. 3);

Sequence ID No. 14 is the amino acid sequence of the C-terminus of BMP-4(FIG. 3);

Sequence ID No. 15 is the amino acid sequence of the C-terminus of DPP(FIG. 3);

Sequence ID No. 16 is the amino acid sequence of the C-terminus of BMP-3(FIG. 3);

Sequence ID No. 17 is the amino acid sequence of the C-terminus of MIS(FIG. 3);

Sequence ID No. 18 is the amino acid sequence of the C-terminus ofinhibin α (FIG. 3);

Sequence ID No. 19 is the amino acid sequence of the C-terminus ofinhibin βA (FIG. 3);

Sequence ID No. 20 is the amino acid sequence of the C-terminus ofinhibin βB (FIG. 3);

Sequence ID No. 21 is the amino acid sequence of the C-terminus ofTGF-β1 (FIG. 3);

Sequence ID No. 22 is the amino acid sequence of the C-terminus ofTGF-β2 (FIG. 3);

Sequence ID No. 23 is the amino acid sequence of the C-terminus ofTGF-,β3 (FIG. 3):

Sequence ID No. 24 is the amino acid sequence of the C-terminus ofTGF-β4 (FIG. 3);

Sequence ID No. 25 is the amino acid sequence of the C-terminus ofTGF-β5 (FIG. 3); and

Sequence ID No. 26 is the amino acid sequence of human GDF-9 (FIG. 6).

28 1 35 DNA Artificial sequence Primer for PCR 1 ccggaattcg gntggvanvantggrtnrtn kcncc 35 2 33 DNA Artificial sequence Primer for PCR 2ccggaattcr canscrcanc ynbtngynry cat 33 3 1712 DNA Mus musculus CDS(29)..(1351) 3 atgcgttcct tcttagttct tccaagtc atg gca ctt ccc agc aacttc ctg 52 Met Ala Leu Pro Ser Asn Phe Leu 1 5 ttg ggg gtt tgc tgc tttgcc tgg ctg tgt ttt ctt agt agc ctt agc 100 Leu Gly Val Cys Cys Phe AlaTrp Leu Cys Phe Leu Ser Ser Leu Ser 10 15 20 tct cag gct tct act gaa gaatcc cag agt gga gcc agt gaa aat gtg 148 Ser Gln Ala Ser Thr Glu Glu SerGln Ser Gly Ala Ser Glu Asn Val 25 30 35 40 gag tct gag gca gac ccc tggtcc ttg ctg ctg cct gta gat ggg act 196 Glu Ser Glu Ala Asp Pro Trp SerLeu Leu Leu Pro Val Asp Gly Thr 45 50 55 gac agg tct ggc ctc ttg ccc cccctc ttt aag gtt cta tct gat agg 244 Asp Arg Ser Gly Leu Leu Pro Pro LeuPhe Lys Val Leu Ser Asp Arg 60 65 70 cga ggt gag acc cct aag ctg cag cctgac tcc aga gca ctc tac tac 292 Arg Gly Glu Thr Pro Lys Leu Gln Pro AspSer Arg Ala Leu Tyr Tyr 75 80 85 atg aaa aag ctc tat aag acg tat gct accaaa gag ggg gtt ccc aaa 340 Met Lys Lys Leu Tyr Lys Thr Tyr Ala Thr LysGlu Gly Val Pro Lys 90 95 100 ccc agc aga agt cac ctc tac aat acc gtccgg ctc ttc agt ccc tgt 388 Pro Ser Arg Ser His Leu Tyr Asn Thr Val ArgLeu Phe Ser Pro Cys 105 110 115 120 gcc cag caa gag cag gca ccc agc aaccag gtg aca gga ccg ctg ccg 436 Ala Gln Gln Glu Gln Ala Pro Ser Asn GlnVal Thr Gly Pro Leu Pro 125 130 135 atg gtg gac ctg ctg ttt aac ctg gaccgg gtg act gcc atg gaa cac 484 Met Val Asp Leu Leu Phe Asn Leu Asp ArgVal Thr Ala Met Glu His 140 145 150 ttg ctc aaa tcg gtc ttg cta tac actctg aac aac tct gcc tct tcc 532 Leu Leu Lys Ser Val Leu Leu Tyr Thr LeuAsn Asn Ser Ala Ser Ser 155 160 165 tcc tcc act gtg acc tgt atg tgt gacctt gtg gta aag gag gcc atg 580 Ser Ser Thr Val Thr Cys Met Cys Asp LeuVal Val Lys Glu Ala Met 170 175 180 tct tct ggc agg gca ccc cca aga gcaccg tac tca ttc acc ctg aag 628 Ser Ser Gly Arg Ala Pro Pro Arg Ala ProTyr Ser Phe Thr Leu Lys 185 190 195 200 aaa cac aga tgg att gag att gatgtg acc tcc ctc ctt cag ccc cta 676 Lys His Arg Trp Ile Glu Ile Asp ValThr Ser Leu Leu Gln Pro Leu 205 210 215 gtg acc tcc agc gag agg agc attcac ctg tct gtc aat ttt aca tgc 724 Val Thr Ser Ser Glu Arg Ser Ile HisLeu Ser Val Asn Phe Thr Cys 220 225 230 aca aaa gac cag gtg cca gag gacgga gtg ttt agc atg cct ctc tca 772 Thr Lys Asp Gln Val Pro Glu Asp GlyVal Phe Ser Met Pro Leu Ser 235 240 245 gtg cct cct tcc ctc atc ttg tatctc aac gac aca agc acc cag gcc 820 Val Pro Pro Ser Leu Ile Leu Tyr LeuAsn Asp Thr Ser Thr Gln Ala 250 255 260 tac cac tct tgg cag tct ctt cagtcc acc tgg agg cct tta cag cat 868 Tyr His Ser Trp Gln Ser Leu Gln SerThr Trp Arg Pro Leu Gln His 265 270 275 280 ccc ggc cag gcc ggt gtg gctgcc cgt ccc gtg aaa gag gaa gct act 916 Pro Gly Gln Ala Gly Val Ala AlaArg Pro Val Lys Glu Glu Ala Thr 285 290 295 gag gtg gaa aga tct ccc cggcgc cgt cga ggg cag aaa gcc atc cgc 964 Glu Val Glu Arg Ser Pro Arg ArgArg Arg Gly Gln Lys Ala Ile Arg 300 305 310 tcc gaa gcg aag ggg cca cttctt aca gca tcc ttc aac ctc agc gaa 1012 Ser Glu Ala Lys Gly Pro Leu LeuThr Ala Ser Phe Asn Leu Ser Glu 315 320 325 tac ttc aaa cag ttt ctt ttcccc caa aac gag tgt gaa ctc cat gac 1060 Tyr Phe Lys Gln Phe Leu Phe ProGln Asn Glu Cys Glu Leu His Asp 330 335 340 ttc aga ctg agt ttt agt cagctc aaa tgg gac aac tgg atc gtg gcc 1108 Phe Arg Leu Ser Phe Ser Gln LeuLys Trp Asp Asn Trp Ile Val Ala 345 350 355 360 ccg cac agg tac aac cctagg tac tgt aaa ggg gac tgt cct agg gcg 1156 Pro His Arg Tyr Asn Pro ArgTyr Cys Lys Gly Asp Cys Pro Arg Ala 365 370 375 gtc agg cat cgg tat ggctct cct gtg cac acc atg gtc cag aat ata 1204 Val Arg His Arg Tyr Gly SerPro Val His Thr Met Val Gln Asn Ile 380 385 390 atc tat gag aag ctg gaccct tca gtg cca agg cct tcg tgt gtg ccg 1252 Ile Tyr Glu Lys Leu Asp ProSer Val Pro Arg Pro Ser Cys Val Pro 395 400 405 ggc aag tac agc ccc ctgagt gtg ttg acc att gaa ccc gac ggc tcc 1300 Gly Lys Tyr Ser Pro Leu SerVal Leu Thr Ile Glu Pro Asp Gly Ser 410 415 420 atc gct tac aaa gag tacgaa gac atg ata gct acg agg tgc acc tgt 1348 Ile Ala Tyr Lys Glu Tyr GluAsp Met Ile Ala Thr Arg Cys Thr Cys 425 430 435 440 cgt tagcatgggggccacttcaa caagcctgcc tggcagagca atgctgtggg 1401 Arg ccttagagtgcctgggcaga gagcttcctg tgaccagtct ctccgtgctg ctcagtgcac 1461 actgtgtgagcgggggaagt gtgtgtgtgt ggatgagcac atcgagtgca gtgtccgtag 1521 gtgtaaagggcacactcact ggtcgttgcc ataaaccaag tgaaatgtaa ctcatttgga 1581 gagctctttctccccacgag tgtagttttc agtggacaga tttgttagca taagtctcga 1641 gtagaatgtagctgtgaaca tgtcagagtg ctgtggtttt atgtgacgga agaataaact 1701 gttgatggca t1712 4 441 PRT Mus musculus 4 Met Ala Leu Pro Ser Asn Phe Leu Leu GlyVal Cys Cys Phe Ala Trp 1 5 10 15 Leu Cys Phe Leu Ser Ser Leu Ser SerGln Ala Ser Thr Glu Glu Ser 20 25 30 Gln Ser Gly Ala Ser Glu Asn Val GluSer Glu Ala Asp Pro Trp Ser 35 40 45 Leu Leu Leu Pro Val Asp Gly Thr AspArg Ser Gly Leu Leu Pro Pro 50 55 60 Leu Phe Lys Val Leu Ser Asp Arg ArgGly Glu Thr Pro Lys Leu Gln 65 70 75 80 Pro Asp Ser Arg Ala Leu Tyr TyrMet Lys Lys Leu Tyr Lys Thr Tyr 85 90 95 Ala Thr Lys Glu Gly Val Pro LysPro Ser Arg Ser His Leu Tyr Asn 100 105 110 Thr Val Arg Leu Phe Ser ProCys Ala Gln Gln Glu Gln Ala Pro Ser 115 120 125 Asn Gln Val Thr Gly ProLeu Pro Met Val Asp Leu Leu Phe Asn Leu 130 135 140 Asp Arg Val Thr AlaMet Glu His Leu Leu Lys Ser Val Leu Leu Tyr 145 150 155 160 Thr Leu AsnAsn Ser Ala Ser Ser Ser Ser Thr Val Thr Cys Met Cys 165 170 175 Asp LeuVal Val Lys Glu Ala Met Ser Ser Gly Arg Ala Pro Pro Arg 180 185 190 AlaPro Tyr Ser Phe Thr Leu Lys Lys His Arg Trp Ile Glu Ile Asp 195 200 205Val Thr Ser Leu Leu Gln Pro Leu Val Thr Ser Ser Glu Arg Ser Ile 210 215220 His Leu Ser Val Asn Phe Thr Cys Thr Lys Asp Gln Val Pro Glu Asp 225230 235 240 Gly Val Phe Ser Met Pro Leu Ser Val Pro Pro Ser Leu Ile LeuTyr 245 250 255 Leu Asn Asp Thr Ser Thr Gln Ala Tyr His Ser Trp Gln SerLeu Gln 260 265 270 Ser Thr Trp Arg Pro Leu Gln His Pro Gly Gln Ala GlyVal Ala Ala 275 280 285 Arg Pro Val Lys Glu Glu Ala Thr Glu Val Glu ArgSer Pro Arg Arg 290 295 300 Arg Arg Gly Gln Lys Ala Ile Arg Ser Glu AlaLys Gly Pro Leu Leu 305 310 315 320 Thr Ala Ser Phe Asn Leu Ser Glu TyrPhe Lys Gln Phe Leu Phe Pro 325 330 335 Gln Asn Glu Cys Glu Leu His AspPhe Arg Leu Ser Phe Ser Gln Leu 340 345 350 Lys Trp Asp Asn Trp Ile ValAla Pro His Arg Tyr Asn Pro Arg Tyr 355 360 365 Cys Lys Gly Asp Cys ProArg Ala Val Arg His Arg Tyr Gly Ser Pro 370 375 380 Val His Thr Met ValGln Asn Ile Ile Tyr Glu Lys Leu Asp Pro Ser 385 390 395 400 Val Pro ArgPro Ser Cys Val Pro Gly Lys Tyr Ser Pro Leu Ser Val 405 410 415 Leu ThrIle Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp 420 425 430 MetIle Ala Thr Arg Cys Thr Cys Arg 435 440 5 117 PRT Homo sapiens 5 Lys ArgArg Ala Ala Ile Ser Val Pro Lys Gly Phe Cys Arg Asn Phe 1 5 10 15 CysHis Arg His Gln Leu Phe Ile Asn Phe Gln Asp Leu Gly Trp His 20 25 30 LysTrp Val Ile Ala Pro Lys Gly Phe Met Ala Asn Tyr Cys His Gly 35 40 45 GluCys Pro Phe Ser Met Thr Thr Tyr Leu Asn Ser Ser Asn Tyr Ala 50 55 60 PheMet Gln Ala Leu Met His Met Ala Asp Pro Lys Val Pro Lys Ala 65 70 75 80Val Cys Val Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp 85 90 95Ser Asp Lys Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val Asp 100 105110 Glu Cys Gly Cys Gly 115 6 118 PRT Homo sapiens 6 Phe Asn Leu Ser GluTyr Phe Lys Gln Phe Leu Phe Pro Gln Asn Glu 1 5 10 15 Cys Glu Leu HisAsp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp Asp 20 25 30 Asn Trp Ile ValAla Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys Gly 35 40 45 Asp Cys Pro ArgAla Val Arg His Arg Tyr Gly Ser Pro Val His Thr 50 55 60 Met Val Gln AsnIle Ile Tyr Glu Lys Leu Asp Pro Ser Val Pro Arg 65 70 75 80 Pro Ser CysVal Pro Gly Lys Tyr Ser Pro Leu Ser Val Leu Thr Ile 85 90 95 Glu Pro AspGly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile Ala 100 105 110 Thr ArgCys Thr Cys Arg 115 7 122 PRT Homo sapiens 7 Pro Arg Arg Asp Ala Glu ProVal Leu Gly Gly Gly Pro Gly Gly Ala 1 5 10 15 Cys Arg Ala Arg Arg LeuTyr Val Ser Phe Arg Glu Val Gly Trp His 20 25 30 Arg Trp Val Ile Ala ProArg Gly Phe Leu Ala Asn Tyr Cys Gln Gly 35 40 45 Gln Cys Ala Leu Pro ValAla Leu Ser Gly Ser Gly Gly Pro Pro Ala 50 55 60 Leu Asn His Ala Val LeuArg Ala Leu Met His Ala Ala Ala Pro Gly 65 70 75 80 Ala Ala Asp Leu ProCys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 85 90 95 Val Leu Phe Phe AspAsn Ser Asp Asn Val Val Leu Arg Gln Tyr Glu 100 105 110 Asp Met Val ValAsp Glu Cys Gly Cys Arg 115 120 8 118 PRT Xenopus sp. 8 Arg Arg Lys ArgSer Tyr Ser Lys Leu Pro Phe Thr Ala Ser Asn Ile 1 5 10 15 Cys Lys LysArg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gln 20 25 30 Asn Trp ValIle Ala Pro Gln Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 35 40 45 Glu Cys ProTyr Pro Leu Thr Glu Ile Leu Asn Gly Ser Asn His Ala 50 55 60 Ile Leu GlnThr Leu Val His Ser Ile Glu Pro Glu Asp Ile Pro Leu 65 70 75 80 Pro CysCys Val Pro Thr Lys Met Ser Pro Ile Ser Met Leu Phe Tyr 85 90 95 Asp AsnAsn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 100 105 110 AspGlu Cys Gly Cys Arg 115 9 118 PRT Mus musculus 9 Arg Val Ser Ser Ala SerAsp Tyr Asn Ser Ser Glu Leu Lys Thr Ala 1 5 10 15 Cys Arg Lys His GluLeu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln 20 25 30 Asp Trp Ile Ile AlaPro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 35 40 45 Glu Cys Ser Phe ProLeu Asn Ala His Met Asn Ala Thr Asn His Ala 50 55 60 Ile Val Gln Thr LeuVal His Leu Met Asn Pro Glu Tyr Val Pro Lys 65 70 75 80 Pro Cys Cys AlaPro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 85 90 95 Asp Asp Asn SerAsn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 100 105 110 Arg Ala CysGly Cys His 115 10 118 PRT Homo sapiens 10 Arg Met Ala Asn Val Ala GluAsn Ser Ser Ser Asp Gln Arg Gln Ala 1 5 10 15 Cys Lys Lys His Glu LeuTyr Val Ser Phe Arg Asp Leu Gly Trp Gln 20 25 30 Asp Trp Ile Ile Ala ProGlu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly 35 40 45 Glu Cys Ala Phe Pro LeuAsn Ser Tyr Met Asn Ala Thr Asn His Ala 50 55 60 Ile Val Gln Thr Leu ValHis Phe Ile Asn Pro Glu Thr Val Pro Lys 65 70 75 80 Pro Cys Cys Ala ProThr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe 85 90 95 Asp Asp Ser Ser AsnVal Ile Leu Lys Lys Tyr Arg Asn Met Val Val 100 105 110 Arg Ala Cys GlyCys His 115 11 118 PRT Homo sapiens 11 Arg Met Ser Ser Val Gly Asp TyrAsn Thr Ser Glu Gln Lys Gln Ala 1 5 10 15 Cys Lys Lys His Glu Leu TyrVal Ser Phe Arg Asp Leu Gly Trp Gln 20 25 30 Asp Trp Ile Ile Ala Pro GluGly Tyr Ala Ala Phe Tyr Cys Asp Gly 35 40 45 Glu Cys Ser Phe Pro Leu AsnAla His Met Asn Ala Thr Asn His Ala 50 55 60 Ile Val Gln Thr Leu Val HisLeu Met Phe Pro Asp His Val Pro Lys 65 70 75 80 Pro Cys Cys Ala Pro ThrLys Leu Asn Ala Ile Ser Val Leu Tyr Phe 85 90 95 Asp Asp Ser Ser Asn ValIle Leu Lys Lys Tyr Arg Asn Met Val Val 100 105 110 Arg Ser Cys Gly CysHis 115 12 118 PRT Drosophila melanogaster 12 Ser Pro Asn Asn Val ProLeu Leu Glu Pro Met Glu Ser Thr Arg Ser 1 5 10 15 Cys Gln Met Gln ThrLeu Tyr Ile Asp Phe Lys Asp Leu Gly Trp His 20 25 30 Asp Trp Ile Ile AlaPro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser Gly 35 40 45 Glu Cys Asn Phe ProLeu Asn Ala His Met Asn Ala Thr Asn His Ala 50 55 60 Ile Val Gln Thr LeuVal His Leu Leu Glu Pro Lys Lys Val Pro Lys 65 70 75 80 Pro Cys Cys AlaPro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr His 85 90 95 Leu Asn Asp GluAsn Val Asn Leu Lys Lys Tyr Arg Asn Met Ile Val 100 105 110 Lys Ser CysGly Cys His 115 13 117 PRT Homo sapiens 13 Glu Lys Arg Gln Ala Lys HisLys Gln Arg Lys Arg Leu Lys Ser Ser 1 5 10 15 Cys Lys Arg His Pro LeuTyr Val Asp Phe Ser Asp Val Gly Trp Asn 20 25 30 Asp Trp Ile Val Ala ProPro Gly Tyr His Ala Phe Tyr Cys His Gly 35 40 45 Glu Cys Pro Phe Pro LeuAla Asp His Leu Asn Ser Thr Asn His Ala 50 55 60 Ile Val Gln Thr Leu ValAsn Ser Val Asn Ser Lys Ile Pro Lys Ala 65 70 75 80 Cys Cys Val Pro ThrGlu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 85 90 95 Glu Asn Glu Lys ValVal Leu Lys Asn Tyr Gln Asp Met Val Val Glu 100 105 110 Gly Cys Gly CysArg 115 14 117 PRT Homo sapiens 14 Arg Ser Pro Lys His His Ser Gln ArgAla Arg Lys Lys Asn Lys Asn 1 5 10 15 Cys Arg Arg His Ser Leu Tyr ValAsp Phe Ser Asp Val Gly Trp Asn 20 25 30 Asp Trp Ile Val Ala Pro Pro GlyTyr Gln Ala Phe Tyr Cys His Gly 35 40 45 Asp Cys Pro Phe Pro Leu Ala AspHis Leu Asn Ser Thr Asn His Ala 50 55 60 Ile Val Gln Thr Leu Val Asn SerVal Asn Ser Ser Ile Pro Lys Ala 65 70 75 80 Cys Cys Val Pro Thr Glu LeuSer Ala Ile Ser Met Leu Tyr Leu Asp 85 90 95 Glu Tyr Asp Lys Val Val LeuLys Asn Tyr Gln Glu Met Val Val Glu 100 105 110 Gly Cys Gly Cys Arg 11515 118 PRT Drosophila melanogaster 15 Lys Arg His Ala Arg Arg Pro ThrArg Arg Lys Asn His Asp Asp Thr 1 5 10 15 Cys Arg Arg His Ser Leu TyrVal Asp Phe Ser Asp Val Gly Trp Asp 20 25 30 Asp Trp Ile Val Ala Pro LeuGly Tyr Asp Ala Tyr Tyr Cys His Gly 35 40 45 Lys Cys Pro Phe Pro Leu AlaAsp His Phe Asn Ser Thr Asn His Ala 50 55 60 Val Val Gln Thr Leu Val AsnAsn Met Asn Pro Gly Lys Val Pro Lys 65 70 75 80 Ala Cys Cys Val Pro ThrGln Leu Asp Ser Val Ala Met Leu Tyr Leu 85 90 95 Asn Asp Gln Ser Thr ValVal Leu Lys Asn Tyr Gln Glu Met Thr Val 100 105 110 Val Gly Cys Gly CysArg 115 16 119 PRT Homo sapiens 16 Gln Thr Leu Lys Lys Ala Arg Arg LysGln Trp Ile Glu Pro Arg Asn 1 5 10 15 Cys Ala Arg Arg Tyr Leu Lys ValAsp Phe Ala Asp Ile Gly Trp Ser 20 25 30 Glu Trp Ile Ile Ser Pro Lys SerPhe Asp Ala Tyr Tyr Cys Ser Gly 35 40 45 Ala Cys Gln Phe Pro Met Pro LysSer Leu Lys Pro Ser Asn His Ala 50 55 60 Thr Ile Gln Ser Ile Val Arg AlaVal Gly Val Val Pro Gly Ile Pro 65 70 75 80 Glu Pro Cys Cys Val Pro GluLys Met Ser Ser Leu Ser Ile Leu Phe 85 90 95 Phe Asp Glu Asn Lys Asn ValVal Leu Lys Val Tyr Pro Asn Met Thr 100 105 110 Val Glu Ser Cys Ala CysArg 115 17 115 PRT Homo sapiens 17 Pro Gly Arg Ala Gln Arg Ser Ala GlyAla Thr Ala Ala Asp Gly Pro 1 5 10 15 Cys Ala Leu Arg Glu Leu Ser ValAsp Leu Arg Ala Glu Arg Ser Val 20 25 30 Leu Ile Pro Glu Thr Tyr Gln AlaAsn Asn Cys Gln Gly Val Cys Gly 35 40 45 Trp Pro Gln Ser Asp Arg Asn ProArg Tyr Gly Asn His Val Val Leu 50 55 60 Leu Leu Lys Met Gln Ala Arg GlyAla Ala Leu Ala Arg Pro Pro Cys 65 70 75 80 Cys Val Pro Thr Ala Tyr AlaGly Lys Leu Leu Ile Ser Leu Ser Glu 85 90 95 Glu Arg Ile Ser Ala His HisVal Pro Asn Met Val Ala Thr Glu Cys 100 105 110 Gly Cys Arg 115 18 121PRT Homo sapiens 18 Leu Arg Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala AlaHis Ala Asn 1 5 10 15 Cys His Arg Val Ala Leu Asn Ile Ser Phe Gln GluLeu Gly Trp Glu 20 25 30 Arg Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe HisTyr Cys His Gly 35 40 45 Gly Cys Gly Leu His Ile Pro Pro Asn Leu Ser LeuPro Val Pro Gly 50 55 60 Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu LeuPro Gly Ala Gln 65 70 75 80 Pro Cys Cys Ala Ala Leu Pro Gly Thr Met ArgPro Leu His Val Arg 85 90 95 Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys TyrGlu Thr Val Pro Asn 100 105 110 Leu Leu Thr Gln His Cys Ala Cys Ile 115120 19 121 PRT Homo sapiens 19 Arg Arg Arg Arg Arg Gly Leu Glu Cys AspGly Lys Val Asn Ile Cys 1 5 10 15 Cys Lys Lys Gln Phe Phe Val Ser PheLys Asp Ile Gly Trp Asn Asp 20 25 30 Trp Ile Ile Ala Pro Ser Gly Tyr HisAla Asn Tyr Cys Glu Gly Glu 35 40 45 Cys Pro Ser His Ile Ala Gly Thr SerGly Ser Ser Leu Ser Phe His 50 55 60 Ser Thr Val Ile Asn His Tyr Arg MetArg Gly His Ser Pro Phe Ala 65 70 75 80 Asn Leu Lys Ser Cys Cys Val ProThr Lys Leu Arg Pro Met Ser Met 85 90 95 Leu Tyr Tyr Asp Asp Gly Gln AsnIle Ile Lys Lys Asp Ile Gln Asn 100 105 110 Met Ile Val Glu Glu Cys GlyCys Ser 115 120 20 120 PRT Homo sapiens 20 Arg Ile Arg Lys Arg Gly LeuGlu Cys Asp Gly Arg Thr Asn Leu Cys 1 5 10 15 Cys Arg Gln Gln Phe PheIle Asp Phe Arg Leu Ile Gly Trp Asn Asp 20 25 30 Trp Ile Ile Ala Pro ThrGly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser 35 40 45 Cys Pro Ala Tyr Leu AlaGly Val Pro Gly Ser Ala Ser Ser Phe His 50 55 60 Thr Ala Val Val Asn GlnTyr Arg Met Arg Gly Leu Asn Pro Gly Thr 65 70 75 80 Val Asn Ser Cys CysIle Pro Thr Lys Leu Ser Thr Met Ser Met Leu 85 90 95 Tyr Phe Asp Asp GluTyr Asn Ile Val Lys Arg Asp Val Pro Asn Met 100 105 110 Ile Val Glu GluCys Gly Cys Ala 115 120 21 114 PRT Homo sapiens 21 Arg Arg Ala Leu AspThr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn 1 5 10 15 Cys Cys Val ArgGln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp 20 25 30 Lys Trp Ile HisGlu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly 35 40 45 Pro Cys Pro TyrIle Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu 50 55 60 Ala Leu Tyr AsnGln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys 65 70 75 80 Val Pro GlnAla Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg 85 90 95 Lys Pro LysVal Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys 100 105 110 Cys Ser22 114 PRT Homo sapiens 22 Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe ArgAsn Val Gln Asp Asn 1 5 10 15 Cys Cys Leu Arg Pro Leu Tyr Ile Asp PheLys Arg Asp Leu Gly Trp 20 25 30 Lys Trp Ile His Glu Pro Lys Gly Tyr AsnAla Asn Phe Cys Ala Gly 35 40 45 Ala Cys Pro Tyr Leu Trp Ser Ser Asp ThrGln His Ser Arg Val Leu 50 55 60 Ser Leu Tyr Asn Thr Ile Asn Pro Glu AlaSer Ala Ser Pro Cys Cys 65 70 75 80 Val Ser Gln Asp Leu Glu Pro Leu ThrIle Leu Tyr Tyr Ile Gly Lys 85 90 95 Thr Pro Lys Ile Glu Gln Leu Ser AsnMet Ile Val Lys Ser Cys Lys 100 105 110 Cys Ser 23 114 PRT Homo sapiens23 Lys Arg Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn 1 510 15 Cys Cys Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp 2025 30 Lys Trp Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly 3540 45 Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu 5055 60 Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 6570 75 80 Val Pro Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg85 90 95 Thr Pro Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys100 105 110 Cys Ser 24 116 PRT Homo sapiens 24 Arg Arg Asp Leu Asp ThrAsp Tyr Cys Phe Gly Pro Gly Thr Asp Glu 1 5 10 15 Lys Asn Cys Cys ValArg Pro Leu Tyr Ile Asp Phe Arg Lys Asp Leu 20 25 30 Gln Trp Lys Trp IleHis Glu Pro Lys Gly Tyr Met Ala Asn Phe Cys 35 40 45 Met Gly Pro Cys ProTyr Ile Trp Ser Ala Asp Thr Gln Tyr Thr Lys 50 55 60 Val Leu Ala Leu TyrAsn Gln His Asn Pro Gly Ala Ser Ala Ala Pro 65 70 75 80 Cys Cys Val ProGln Thr Leu Asp Pro Leu Pro Ile Ile Tyr Tyr Val 85 90 95 Gly Arg Asn ValArg Val Glu Gln Leu Ser Asn Met Val Val Arg Ala 100 105 110 Cys Lys CysSer 115 25 114 PRT Homo sapiens 25 Lys Arg Gly Val Gly Gln Glu Tyr CysPhe Gly Asn Asn Gly Pro Asn 1 5 10 15 Cys Cys Val Lys Pro Leu Tyr IleAsn Phe Arg Lys Asp Leu Gly Trp 20 25 30 Lys Trp Ile His Glu Pro Lys GlyTyr Glu Ala Asn Tyr Cys Leu Gly 35 40 45 Asn Cys Pro Tyr Ile Trp Ser MetAsp Thr Gln Tyr Ser Lys Val Leu 50 55 60 Ser Leu Tyr Asn Gln Asn Asn ProGly Ala Ser Ile Ser Pro Cys Cys 65 70 75 80 Val Pro Asp Val Leu Glu ProLeu Pro Ile Ile Tyr Tyr Val Gly Arg 85 90 95 Thr Ala Lys Val Glu Gln LeuSer Asn Met Val Val Arg Ser Cys Asn 100 105 110 Cys Ser 26 454 PRT Homosapiens 26 Met Ala Arg Pro Asn Lys Phe Leu Leu Trp Phe Cys Cys Phe AlaTrp 1 5 10 15 Leu Cys Phe Pro Ile Ser Leu Gly Ser Gln Ala Ser Gly GlyGlu Ala 20 25 30 Gln Ile Ala Ala Ser Ala Glu Leu Glu Ser Gly Ala Met ProTrp Ser 35 40 45 Leu Leu Gln His Ile Asp Glu Arg Asp Arg Ala Gly Leu LeuPro Ala 50 55 60 Leu Phe Lys Val Leu Ser Val Gly Arg Gly Gly Ser Pro ArgLeu Gln 65 70 75 80 Pro Asp Ser Arg Ala Leu His Tyr Met Lys Lys Leu TyrLys Thr Tyr 85 90 95 Ala Thr Lys Glu Gly Ile Pro Lys Ser Asn Arg Ser HisLeu Tyr Asn 100 105 110 Thr Val Arg Leu Phe Thr Pro Cys Thr Arg His LysGln Ala Pro Gly 115 120 125 Asp Gln Val Thr Gly Ile Leu Pro Ser Val GluLeu Leu Phe Asn Leu 130 135 140 Asp Arg Ile Thr Thr Val Glu His Leu LeuLys Ser Val Leu Leu Tyr 145 150 155 160 Asn Ile Asn Asn Ser Val Ser PheSer Ser Ala Val Lys Cys Val Cys 165 170 175 Asn Leu Met Ile Lys Glu ProLys Ser Ser Ser Arg Thr Leu Gly Arg 180 185 190 Ala Pro Tyr Ser Phe ThrPhe Asn Ser Gln Phe Glu Phe Gly Lys Lys 195 200 205 His Lys Trp Ile GlnIle Asp Val Thr Ser Leu Leu Gln Pro Leu Val 210 215 220 Ala Ser Asn LysArg Ser Ile His Met Ser Ile Asn Phe Thr Cys Met 225 230 235 240 Lys AspGln Leu Glu His Pro Ser Ala Gln Asn Gly Leu Phe Asn Met 245 250 255 ThrLeu Val Ser Pro Ser Leu Ile Leu Tyr Leu Asn Asp Thr Ser Ala 260 265 270Gln Ala Tyr His Ser Trp Tyr Ser Leu His Tyr Lys Arg Arg Pro Ser 275 280285 Gln Gly Pro Asp Gln Glu Arg Ser Leu Ser Ala Tyr Pro Val Gly Glu 290295 300 Glu Ala Ala Glu Asp Gly Arg Ser Ser His His Arg His Arg Arg Gly305 310 315 320 Gln Glu Thr Val Ser Ser Glu Leu Lys Lys Pro Leu Gly ProAla Ser 325 330 335 Phe Asn Leu Ser Glu Tyr Phe Arg Gln Phe Leu Leu ProGln Asn Glu 340 345 350 Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser GlnLeu Lys Trp Asp 355 360 365 Asn Trp Ile Val Ala Pro His Arg Tyr Asn ProArg Tyr Cys Lys Gly 370 375 380 Asp Cys Pro Arg Ala Val Gly His Arg TyrGly Ser Pro Val His Thr 385 390 395 400 Met Val Gln Asn Ile Ile Tyr GluLys Leu Asp Ser Ser Val Pro Arg 405 410 415 Pro Ser Cys Val Pro Ala LysTyr Ser Pro Leu Ser Val Leu Thr Ile 420 425 430 Glu Pro Asp Gly Ser IleAla Tyr Lys Glu Tyr Glu Asp Met Ile Ala 435 440 445 Thr Lys Cys Thr CysArg 450 27 4 PRT Mus musculus 27 Arg Arg Arg Arg 1 28 4 PRT Artificialsequence Putative proteolytic cleavege site 28 Arg Xaa Xaa Arg

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: a) SEQ ID NO:3; b) SEQ ID NO:3, wherein T can alsobe U; c) nucleic acid sequences complementary to a nucleic acid sequenceencoding SEQ ID NO: 4 or SEQ ID NO:26; d) fragments of any of a) throughc) that are at least 15 bases in length and that will hybridize to DNAwhich encodes the GDF-9 protein of SEQ ID NO:4 or SEQ ID NO:26; e)degenerate variants of any of a), b) and d); and f) nucleic acidsequences complementary to a degenerate variant of e).
 2. An isolatedpolynucleotide encoding a growth differentiation factor-9 polypeptide,wherein the polynucleotide is isolated from a mammalian cell.
 3. Thepolynucleotide of claim 2, wherein the mammalian cell is selected fromthe group consisting of a mouse, a rat, and a human cell.
 4. Anexpression vector including a polynucleotide of claim
 2. 5. The vectorof claim 4, wherein the vector is a plasmid.
 6. The vector of claim 4,wherein the vector is a viral vector.
 7. A host cell containing thevector of claim
 4. 8. The host cell of claim 7, wherein the cell isprokaryotic.
 9. The host cell of claim 7, wherein the cell iseukaryotic.